Escherichia coli O157:H7 is an exceptionally virulent food-borne, human pathogen that causes a spectrum of illness, including asymptomatic and post-symptomatic carriage, mild diarrhea, bloody diarrhea/hemorrhagic colitis, and the postdiarrheal, potentially lethal, hemolytic uremic syndrome (HUS). (Wilson et al., J Infect Dis, 174:1021-1027 (1996); (Karch et al., J Clin Microbiol, 33:1602-1605 (1995); (Rodrigue et al., J Infect Dis, 172:1122-1125 (1995); (Riley et al., N Engl J Med, 308:681-685 (1983); (Karmali et al., Lancet, 1:619-620 (1983); Neill et al., Arch Intern Med, 145:2215-2217 (1985); Neill et al., Pediatrics, 80:37-40 (1987); and Tarr et al., J Infect Dis, 162:553-556 (1990)). While other E. coli strains are considered in some contexts to be pathogens, the excessive pathogenicity of E. coli O157:H7 is a well recognized distinguishing feature.
HUS is defined as a triad of non-immune microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. HUS is chiefly a disorder of children under age 10, however, the elderly are also susceptible to severe complications of E. coli O157:H7 gastrointestinal infections. (Martin et al., N Engl J Med, 323:1161-1167 (1990); Siegler et al., Pediatrics, 94:35-40 (1994); Tarr and Hickman, Pediatrics, 80:41-45 (1987); Tarr et al., Am J Epidemiol, 129:582-586 (1989); Tarr et al., J Infect Dis, 162:553-556 (1990); (Carter et al., N Engl J Med. 317:1496-1500 (1987); and Ryan et al., J Infect Dis, 154:631-638 (1986)).
HUS follows gastrointestinal infection with E. coli O157:H7 in approximately 10-15% of pediatric cases. (Bell et al., JAMA, 272:1349-1353 (1994) and Bell et al., Pediatrics, 100:E12 (1997)). Approximately three-quarters of children with HUS require blood transfusions and approximately one-half require dialysis. (Tarr et al., Am J Epidemiol, 129:582-586 (1989); (Brandt et al., J Pediatr, 125:519-526 (1994); and Tarr et al., Am J Epidemiol, 129:582-586 (1989)). Despite recognition of O157:H7 infection and the use of modern pediatric intensive care, about 5-10% of those infected die. (Brandt et al., J Pediatr, 125:519-526 (1994) and Tarr et al., Am J Epidemiol, 129:582-586 (1989)). Investigation of O157:H7 outbreaks have provided evidence that the infectious dose is low. For example, limited exposure to a municipal lake in Portland, Oreg., wherein the levels of E. coli O157:H7 were undetectable, was sufficient to produce disease in visitors. (Keene et al., N Engl J Med, 331:579-584 (1994)) and during a salami-associated outbreak in the Pacific Northwest in 1994, investigators concluded that the people who became ill had consumed between 2 and 45 viable E. coli O157:H7 organisms. (Tilden et al., Am J Public Health, 86:1142-1145 (1996)).
E. coli O157:H7 is often found in food and environmental vehicles that do not always undergo an efficient bacterial killing process. Large outbreaks have been caused by the interstate dissemination of contaminated ground beef that was under cooked (Bell et al., JAMA, 272:1349-1353 (1994) and Riley et al., N Engl J Med, 308:681-685 (1983)); salted, fermented, but uncooked salami (Tilden et al., Am J Public Health, 86:1142-1145 (1996)); municipal (Swerdlow et al., Ann Intern Med, 117:812-819 (1992)) and swimming (Keene et al., N Engl J Med, 331:579-584 (1994)) water; unpasteurized apple juice (Anonymous, Morb Mortal Wkly Rep, 45:975 (1996)); unpasteurized milk (Keene et al., J Infect Dis, 176:815-818 (1997)); and lettuce (Ackers et al., J Infect Dis, 177:1588-1593 (1998)). Improper food handling has been reported to be a significant factor associated with human infection. (Mead et al., Arch Intern Med, 157:204-208 (1997)).
E. coli O157:H7 has not been shown to possess a capsular polysaccharide but it expresses an O side chain antigen designated 157, which consists of repeating tetrasaccharide units of variable length. These tetrasaccharide units comprise the antigenic O157 lipopolysaccaride (LPS). In contrast to other E. coli strains, O157:H7 fails to ferment sorbitol after overnight culture on MacConkey agar into which sorbitol rather than lactose is incorporated as the carbon source. (Wells et al., J Clin Microbiol, 18:512-520 (1983); March et al., J Clin Microbiol, 23:869-872 (1986)). E. coli O157:H7 also fails to produce β-glucuronidase, another metabolic distinguishing factor. (Ratnam et al., J Clin Microbiol, 26:2006-2012 (1988)). Sorbitol non-fermenting E. coli almost always express the H7 flagellar antigen, though occasional sorbitol non-fermenting E. coli O157 strains recovered in the United States do not express the H7 antigen. (Slutsker et al., Ann Intern Med, 126:505-513 (1997)). Another variant of E. coli O157:H7 has been found in Germany and Czech Republic, which expresses the O157 antigen, but are non-motile pathogens that ferment sorbitol. (Bielaszewska et al., J Clin Microbiol, 36:2135-2137 (1998); Gunzer et al., J Clin Microbiol, 30:1807-1810 (1992)). Such sorbitol non-fermenting E. coli O157 variants are difficult to identify by using the sorbitol MacConkey agar screening technique.
Current diagnostic approaches involve monitoring the growth characteristics of cultured E. coli on MacConkey agar, as described above, and utilizing a seriological agent specific for O157 LPS. That is, organisms with an appearance typical of E. coli on sorbitol MacConkey agar, that fail to ferment sorbitol, react with a serologic reagent specific for the O157 LPS side chain but fail to react with a control (negative) reagent are considered to be Shiga-toxigenic, and, presumably, pathogenic, E. coli O157:H7. The identification of the H7 antigen and the toxinogenic phenotype are not necessary for clinical purposes because sorbitol non-fermenting E. coli that are non mucoid, react with a specific O157 antigen determining reagent and do not react with a negative control reagent are almost always toxigenic. (Strockbine et al., “Overview of detection and subtyping methods,” Escherichia coli O157:H7 and other Shiga toxin-producing E. coli, Chapter 33, Kaper and O'Brien, eds., Washington, D.C.: ASM Press, 1998:331-356 and Tarr, “Shiga toxin-producing Escherichia coli infections: challenges and opportunities,” Escherichia coli O157:H7 and other Shiga toxin-producing E. coli, Chapter 39, Kaper and O'Brien, eds., Washington, D.C.: ASM Press, 1998:393-402).
Alternate diagnostic approaches have been recently developed. One approach involves the detection of the presence of released Shiga-toxin. These tests either exploit the ability of Shiga-toxins to bind to a glycosphingolipid ligand (globotriaosylceramide) (Basta et al., J Clin Microbiol, 27:1617-1622 (1989)) (Biocarb, Gaithersburg, Md.) or employ an enzyme immunoassay (Meridian Diagnostics, Cincinnati, Ohio). (Kehl et al., J Clin Microbiol, 35:2051-2054 (1997)); Park et al., Diag Microbiol Infect Dis, 26:69-72 (1996)). These tests have the advantage that they detect Shiga toxigenic E. coli besides E. coli O157:H7. Several diagnostic tests also involve the use of probes or primers to detect sequences of O157:H7 through hybridization, enzyme cleavage, or Polymerase Chain Reaction (PCR). (See e.g., U.S. Pat. Nos. 5,738,995; 5,747,257; and 5,756,293).
A variety of techniques to identify excessively pathogenic E. coli in food have also been developed. (Bennett et al., Lett Appl Microbiol, 22:237-243 (1996); Bennett et al., Lett Appl Microbiol, 20:375-379 (1995); Blanco et al., Microbiologia, 12:385-394 (1996); Bolton et al., Lett Appl Microbiol, 23:317-321 (1996); Doyle and Schoeni, Appl Environ Microbiol, 53:2394-2396 (1987); Feldsine et al., JAOAC Int, 80:517-529 (1997); Feldsine et al., JAOAC Int, 80:530-543 (1997); Feldsine et al., J AOAC Int, 80:43-48 (1997); Feldsine et al., J AOAC Int, 80:37-42 (1997); Jinneman et al., J Food Protect, 58:722-726 (1995); Johnson et al., Appl Environ Microbiol, 61:386-388 (1995); Kim and Doyle, Appl Environ Microbiol, 58:1764-1767 (1992); Notermans et al., Int J Food Microbiol, 13:31-40 (1991); Okrend et al., J Food Protect, 53:936-940 (1990); Padhye and Doyle, Appl Environ Microbiol, 57:2693-2698 (1991); Pawelzik, Acta Microbiol Hung, 38:315-320 (1991); Ratnam and March, Can Med Assoc J, 134:43-46 (1986); Read et al., Epidemiol Infect, 105:11-20 (1990); Sequel, Can Med Assoc J, 143:519-521 (1990); Tortorello and Stewart, Appl Environ Microbiol, 60:3553-3559 (1994); Vernozy-Rozand et al., Revue de Medecine Veterinaire, 149:239-244 (1998); Vemozy-Rozand et al., Revue de Medecine Veterinaire, 148:879-882 (1997); Vernozy-Rozand et al., Lett Appl Microbiol, 25:442-446 (1997); Willshaw et al., J Appl Bacteriol, 75:420-426 (1993); Yu and Bruno, Appl Environ Microbiol, 62:587-592 (1996)). Many of these techniques include a hydrophobic grid membrane filter (Doyle and Schoeni, Appl Environ Microbiol, 53:2394-2396 (1987)), a dipstick immunoassay (Padhye and Doyle, Appl Environ Microbiol, 57:2693-2698 (1991)), multiplex polymerase chain reaction (Jinneman et al., J Food Protect, 58:722-726 (1995)), standard microbiologic techniques, immunomagnetic bead separation (Bennett et al., Lett Appl Microbiol, 22:237-243 (1996); Blanco et al., Microbiologia, 12:385-394 (1996); Karch et al., J Clin Microbiol, 34:516-519 (1996); Vernozy-Rozand et al., Lett Appl Microbiol, 25:442-446 (1997); and (Yu and Bruno, Appl Environ Microbiol, 62:587-592 (1996)) or combinations thereof. There remains a need for a better understanding of the origin of virulent strains of E. coli, in particular O157:H7, and novel approaches to rapidly detect the presence of these organisms in infected individuals and vehicles including, but not limited to, food and water supplies.